Antibiotic resistance systems in microorganisms pose a threat to human health. Metals such as silver and copper are used as antimicrobial agents in a variety of settings, notably for silver in burn wound treatment, since intracellular metals levels must be properly maintained for an organism's survival. However, both pathogenic and non-pathogenic microbes have resistance mechanisms to survive under conditions of high environmental metal levels. In gram-negative bacteria, proton-substrate antiporter systems, similar to the multidrug exporter systems, transport copper and silver to the extracellular space. These metal transporters differ from the multidrug systems in that they have a fourth component which is located in the periplasm. The goals of this proposal are to characterize the structure and function of this fourth periplasmic component to understand its role in metal homeostasis. This component, CusF in the Cus system from E. coli, is expected to serve a metallochaperone function or a metal-dependent regulatory function in its interactions with the rest of the efflux complex, CusCBA. To test the hypothesis that CusF functions as a metallochaperone, we will determine whether CusF transfers metal to CusB in vitro. To establish whether CusF play a regulatory role, we will investigate if metal transfer from CusF in vivo is crucial to its function. The proportions of CusF to CusCBA will be determined, as an excess of CusF in proportion to the rest of the complex may indicate a role as a metallochaperone, while a lower stoichiometry may indicate a regulatory function. Additionally, the proteins with which CusF interact in vivo will be identified. Structural and biochemical experiments are proposed to determine the atomic level details of metal and protein-protein interactions of CusF and CusB. The structure of CusF in the metal-bound state will be determined. Crystals of CusB have been recently obtained, and we anticipate that structural details will be forthcoming. Structural information for the CusF/CusB complex and the coordination of metal by this complex will be determined by NMR and EXAFS experiments. The affinities of CusF and CusB and selected mutants for metals and for the protein partner will be measured by isothermal titration calorimetry. Fundamental information pertinent to microbial resistance to metal-based antibiotics will be obtained from the proposed experiments. An understanding of the mechanisms by which microorganisms confer antibiotic resistance will have impact on public health in the future development and use of broad spectrum antibiotics.